Oxidative and carbonyl stress induced AMD and Codonopsis lanceolata ameliorates AMD via controlling oxidative and carbonyl stress

Age-related macular degeneration (AMD) is one of the leading causes of blindness. AMD is currently incurable; the best solution is to prevent its occurrence. To develop drugs for AMD, it is crucial to have a model system that mimics the symptoms and mechanisms in patients. It is most important to develop safer and more effective anti-AMD drug. In this study, the dose of A2E and the intensity of blue light were evaluated to establish an appropriate atrophic in vitro model of AMD and anti-AMD effect and therapeutic mechanism of Codonopsis lanceolata. The experimental groups included a control group an AMD group treated with A2E and blue light, a lutein group treated with 25 μM lutein after AMD induction, and three groups treated with different doses of C. lanceolata (10, 20, and 50 μg/mL) after AMD induction. Intrinsic apoptotic pathway (Bcl-2 family), anti-oxidative system (Keap1/Nrf2/HO-1 antioxidant response element), and anti-carbonyl effect (4-hydroxynonenal [4-HNE]) were evaluated using immunofluorescence, MTT, TUNEL, FACS, and western blotting analyses. A2E accumulation in the cytoplasm of ARPE-19 cells depending on the dose of A2E. Cell viability of ARPE-19 cells according to the dose of A2E and/or blue light intensity. The population of apoptotic or necrotic cells increased based on the A2E dose and blue light intensity. Codonopsis lanceolata dose-dependently prevented cell death which was induced by A2E and blue light. The antiapoptotic effect of that was caused by activating Keap1/Nrf2/HO-1 pathway, suppressing 4-HNE, and modulating Bcl-2 family proteins like increase of antiapoptotic proteins such as Bcl-2 and Bcl-XL and decrease of proapoptotic protein such as Bim. Based on these findings, 30 μM A2E and 20 mW/cm2 blue light on adult retinal pigment epithelium-19 cells was an appropriate condition for AMD model and C. lanceolata shows promise as an anti-AMD agent.


A2E synthesis
A2E was synthesized according to Parish's method 27 .Briefly, all-trans-retinal (100 mg, 352 mM) and 9.5 mg of ethanolamine (155 mM) were dissolved in 3.0 mL of ethanol (EtOH).Then, 9.3 µL of acetic acid (155 mM) was added, and the mixture was reacted at 120 rpm at room temperature in the dark for 48 h (Fig. 1A).To evaluate the purity of the synthetic A2E, liquid chromatography was conducted using a Waters Alliance 2695 HPLC system with a photodiode array detector.The analytic conditions are described in Table 1.

Manufacturing and intensity measurement of blue light transilluminator
The blue light emitting diodes (LEDs, Sumbulbs, 12 V Blue Light, Shenzhen, China) were used for the blue light transilluminator.The light intensity and wavelength were tested using the NeoLight PL5000 (PIMACS, Kyunggi, Korea), the Programmable AC Power Source 61,052 (Chroma ATE Inc., Taoyuan, Taiwan), and the Voltech PM3000A Universal Power Analyzer (Advanced Test Equipment Corp. CA, USA) by Korea Photonic Technology Institute (KOPTI, Gwangju, Korea).The analytical condition was maintained at 25.1 ± 0.6 °C and 51.8 ± 1.5% relative humidity.
To measure the cytotoxicity of synthetic A2E on ARPE-19 cells, the cells were seeded at a density of 2 × 10 3 cells per well.After 36 h, various doses of A2E (0, 10, 20, 30, and 40 μM) were administered.Then, 24 h after A2E treatment, three levels of blue light intensity (0, 20, and 40 mW/cm 2 ) were applied for 15 min.After 24 h, an MTT assay was conducted to evaluate the cytotoxicity of A2E and/or blue light in ARPE-19 cells, and the results were obtained using a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

The analysis of apoptosis pathway of ARPE-19 cells by A2E and/or blue light
The abundance of apoptotic ARPE-19 cells depending on the dose of synthetic A2E and/or the intensity of blue light was measured using IF, TUNEL staining, and FACS analysis.Based on the MTT assay and A2E accumulation, the doses of A2E and the intensity of blue light were confirmed as 0, 30, or 40 μM A2E and 0, 20, or 40 mW/cm 2 blue light.For the FACS analysis, Invitrogen Alex Fluor 488 apoptosis detection kit (Invitrogen, Waltham, MA, USA, R37174) was used.The cells were harvested using trypsin (Gibco, 25,200,056).Single cells were washed, stained using Alex Fluor 488 and propidium iodide from the kit, and the results were acquired using Guava Easycyte (Millipore, Burlington, MA, USA).
For the TUNEL assay, the Click-iT™ Plus TUNEL assay kit (Invitrogen, C10618) was used.A2E cells were treated under the same conditions regarding A2E dose and/or blue light intensity.The media was discarded, and the attached cells were washed with phosphate buffered saline (PBS, Lonza, Walkersville, MD, USA, BE17-517Q).The cells were then fixed with 4% formaldehyde (Daejung, Siheung-si, Gyeonggi-do, Korea, P2031) in PBS.After fixation, 0.25% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA, 9036-19-5) in PBS was added, and the cells were stained following the manufacturer's guidelines.Finally, the images were obtained using a K1-Fluo confocal microscope (Nanoscope systems).

The analysis of carbonyl stress and oxidative stress on ARPE-19 cells by A2E and/or blue light
Carbonyl stress and oxidative stress were assessed using western blotting.To evaluate the carbonyl stress induced by A2E and/or blue light, the representative carbonyl stress-induced metabolite, 4-HNE (abcam, Ab46545), was analyzed.Oxidative stress analysis was conducted using primary antibodies for Nrf2 (Invitrogen, PA5-27,882) and HO-1 (Invitrogen, PA5-77,833).

Preparation and analysis of C. lanceolata extract and active compounds
The root of C. lanceolata was obtained from Wellphyto Co. (Gwangju, Korea) and deposited at Mokpo National University (MNUCSS-CL-01).To extract the active compounds, 20 g of dried C. lanceolata root was subjected to hot water extraction at 100 ℃ for 4 h.The resulting extract was freeze-dried.In our previous study 28 , lobetylin was confirmed as an active compound in C. lanceolata.This study was conducted to comply with the IUCN Policy Statement on Research involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Analysis of the anti-cytotoxic and anti-apoptotic effects of C. lanceolata in an in vitro model of AMD
All in vitro experiments were performed as shown in Fig. 2. To determine the proper concentration of C. lanceolata for this study, five doses of C. lanceolata (5, 10, 25, 50, and 100 μM) were tested, along with a normal control group (CON; 0 μM A2E and 20 mW/cm 2 blue light), an induction control group (A2E; 30 μM A2E and 20 mW/cm 2 blue light), and a positive control group (Lutein; 30 μM A2E, 20 mW/cm 2 blue light, and 25 μM lutein).At 12 h after seeding ARPE-19 cells in a 96-well plate (2 × 10 3 cell/well), the reagents were administered.At 24 h after C. lanceolata treatment, A2E was added to each well, excluding the normal control group.At 24 h after A2E addition, each well was irradiated with 20 mW/cm 2 blue light for 15 min.Finally, 24 h after blue light irradiation, the MTT assay was conducted to evaluate the anti-cytotoxic effect of C. lanceolata.
The anti-apoptotic effect of C. lanceolata was measured using IF for TUNEL staining and FACS analysis.The experimental groups included the CON, A2E, Lutein, and three doses of C. lanceolata (10, 25, 50 μg/mL) www.nature.com/scientificreports/groups.A2E was applied 24 h after seeding the ARPE-19 cells, and blue light was irradiated after 48 h and after 72 h.The cells were harvested using trypsin to evaluate apoptosis.The subsequent steps were the same for IF, TUNEL, and FACS analyses.

Statistics
The results are expressed as the mean ± standard deviation (SD).Following one-way analysis of variance (ANOVA) analysis, Dunnett's multiple comparison was conducted.Differences with p values below 0.05 were considered significant.

A2E synthesis and the light wavelength of a self-manufactured transilluminator
As shown in Fig. 1A, 352 mM all-trans-retinal and 155 mM ethanolamine were dissolved in 3.0 mL of EtOH.Then, 155 mM acetic acid was added to the mixture, and they were reacted through centrifugation at 120 rpm at room temperature in the dark for 48 h (Fig. 1A).After the reaction, three types of A2E were synthesized and verified using liquid chromatographic analysis.One A2E was found approximately at 12 min (retention time), while two A2E isotypes (Iso-A2Es) were found around 14 min and 16 min (Fig. 1B).The analytical conditions are presented in Table 1.The most effective light for AMD induction is blue light, which has a wavelength range of 400-500 nm 29 .The self-manufactured blue light transilluminator used in this study mainly emitted blue light within this range, with a peak of the wavelength of 454.6 nm (Fig. 1C).

A2E permeated into ARPE-19 cells and the population of dead cells increased depending on both A2E and blue light
A2E, when reacted with blue light, induces AMD and exhibits autofluorescence 30 .To evaluate the level of A2E permeation in the cytoplasm, an IF assay was performed after treatment with 0, 10, 20, 30, or 40 μM A2E.In treatments with 10 μM A2E or less, the green color was unclear.However, in treatments with 20 μM A2E or more, green spots could be clearly detected in the cytoplasm (Fig. 3A).A2E decreased cell viability from 100 ± 7.75% in the 0 μM treatment group to 83.4 ± 9.50% in the 40 μM treatment group (Fig. 3B and Table 2) without blue light treatment.Co-treatment with A2E and blue light dose-dependently increased cytotoxicity.Under 20 mW/ cm 2 blue light illumination, the viabilities in the 30 μM and 40 μM A2E treatment groups were 73.5 ± 9.91% and 72.5 ± 8.89%, respectively.Under 40 mW/cm 2 blue light exposure, the viabilities in the 30 μM and 40 μM A2E treatment groups were only 68.1 ± 6.96% and 63.2 ± 7.17%, respectively.Based on these results, the optimal condition was determined to be 30 μM A2E treatment with 20 mW/cm 2 blue light illumination.www.nature.com/scientificreports/

A2E and blue light induces apoptosis in ARPE-19 cells via Nrf2/HO-1 pathway and carbonyl stress
To analyze the apoptotic effect of A2E and/or blue light treatment, TUNEL assay was performed followed by IF analysis (Fig. 4A).The results were similar to those observed in cell viability tests (Fig. 3B).A2E treatment dose-dependently increased the number of apoptotic cells.Under 20 mW/cm 2 blue light illumination, 30 μM or 40 μM A2E treatment induced cell apoptosis, particularly resulting in irregular cell morphology.Similar results were obtained with 40 mW/cm 2 blue light treatment.To confirm cell death caused by A2E and blue light, FACS analysis was conducted (Table 3).A2E treatment alone dose-dependently increased the population of dead cells, including necrotic cells, from about 5.1 ± 0.68% in the 0 μM A2E treatment group to 23.4 ± 3.50% in the 40 μM A2E treatment group.Co-treatment with A2E and blue light also dose-dependently increased the number of dead cells.Specifically, after 30 μM A2E treatment with 20 mW/cm 2 blue light exposure, the population of apoptotic cells was 21.6 ± 0.072%.Accordingly, this condition was selected as the AMD cell model.Although there are many factors that induce cell death, oxidative and carbonyl stress are among the important ones 31 .To examine the effect of treatment with A2E, blue light, or their combination on oxidative and carbonyl stress, the levels of oxidative stress markers, such as the Nrf2/HO-1 pathway and the carbonyl stress marker 4-HNE were measured (Fig. 4B).Without blue light treatment, an upregulation of 4-HNE due to A2E treatment was observed.However, under 20 mW/cm 2 blue light treatment, A2E treatment suppressed the Nrf2/HO-1 pathway but increased the level of 4-HNE in a dose-dependent manner.

C. lanceolata prevented cell death induced by A2E and blue light in a dose-dependent manner
To measure the preventive effect of C. lanceolata against cytotoxicity induced by A2E and blue light, an MTT assay was performed (Fig. 5A).Co-treatment with 30 μM A2E and 20 mW/cm 2 blue light increased the population of dead ARPE-19 cells, but 25 μM lutein treatment prevented their cytotoxicity.Although only in the 100 μg/ mL C. lanceolata treatment group, the live cell percentage was similar to that in the 25 μM lutein treatment group, C. lanceolata treatment resulted an increasing trend of live cell population depending on the dose.A2E and blue light co-treatment significantly induced cell death compared to the control, but C. lanceolata treatment dose-dependently decreased the level of apoptosis (green) caused by A2E and blue light co-treatment (Fig. 4B).
To confirm the result of the IF assay (Fig. 5B), the anti-apoptotic effect of C. lanceolata was analyzed using FACS (Table 4).Compared to the result in the control group (the population of total apoptotic cells, 5.8 ± 2.37%), A2E and blue light co-treatment induced a higher amount of cell death (10.4 ± 2.90%), but C. lanceolata treatment dose-dependently prevented A2E and blue light-induced cell death.In particular, the population of dead cells in the 50 μg/mL C. lanceolata treatment group (5.0 ± 1.83%) was similar to that in the control group.Compared to the population of normal cells in the 25 μM lutein treatment group (82.2 ± 0.95%), the population in the 25 μg/ mL C. lanceolata treatment group was 83.5 ± 2.03%, indicating that the normal cell percentage in the lutein treatment group was similar to that in the 25 μg/mL C. lanceolata treatment group.

C. lanceolata prevented apoptosis in ARPE-19 cells by increasing Bcl-2 and Bcl-XL and decreasing Bim, stimulating the activation of the Keap1/Nrf2/HO-1, and downregulating 4-HNE
The results shown in Fig. 5 suggested that C. lanceolata prevented apoptosis in ARPE-19 cells, which was caused by co-treatment with A2E and blue light.The Bcl-2 family includes anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, as well as the pro-apoptotic protein Bim 32 .We analyzed the Bcl-2 family proteins to investigate the anti-apoptotic mechanism of C. lanceolata (Fig. 6A).Co-treatment with A2E and blue light regulated the levels of Bcl-2 family proteins by downregulating Bcl-2 and Bcl-XL and upregulating Bim.However, C. lanceolata suppressed this effect in a dose-dependent manner, increasing Bcl-2 and Bcl-XL levels while decreasing Bim expression (Fig. 6A).Keap1/ Nrf2/ HO-1 pathway plays an important role in the anti-oxidative system.Under normal conditions, Keap1 and Nrf2 are bound together in the cytoplasm.However, when cells are stressed, Nrf2 separates from Keap1 and translocates to the nucleus, acting as a transcription factor for HO-1 33 .C. lanceolata increased the expression levels of Keap1, Nrf2, and HO-1 proteins in a dose-dependent manner and effectively reduced the level of 4-HNE (Fig. 6B).To confirm the activation of the Keap1/Nrf2/HO-1 pathway, an IF assay was performed (Fig. 6C and D).In the A2E group, the expression level of Nrf2 in the nucleus was lower compared to the control group.However, similar to lutein treatment, C. lanceolata treatment dose-dependently increased the expression of Nrf2 in the nucleus (Fig. 6C).Similarly, the expression of Keap1 and HO-1 were dose-dependently increased in the cytoplasm (Fig. 6D), consistent with the results of western blotting in the C. lanceolata treatment group.Overall, these results indicate that C. lanceolata effectively activated the Keap1/Nrf2/HO-1 pathway.

Discussion
AMD is one of the leading causes of blindness worldwide 1 , and it is closely associated with aging.The global population of aging individuals is rapidly increasing 4 .According to the stage of AMD, medications can be divided into two types: treatments for atrophic and neovascular AMD.The therapeutic method for neovascular AMD includes laser or anti-VEGF treatment, which aims to inhibit angiogenesis in the choroidal area 10 .The purpose of atrophic AMD treatment is to prevent the death of RPE cells and is associated with eliminating various factorsrelated to apoptosis, such as oxidative stress, inflammation, and carbonyl stress.Current medications have various adverse effects such as macular edema, retinovascular disease, hypertension, insulin resistance, insomnia, skin thinning, gastric ulceration, eye bleeding, and increased risk of infection [11][12][13][14] .Due to these adverse effects, there is   www.nature.com/scientificreports/ a need for new AMD drugs based on the type of AMD such as atrophic or neovascular AMD that are both more effective and safer.For successful drug development, it is crucial to establish an appropriate AMD model system.Depending on the time of occurrence and angiogenesis in the choroidal area, AMD can be classified into two types: atrophic and neovascular AMD.The main difference between them is that atrophic AMD is caused by the death of RPE cells in the early stage.While neovascular AMD occurs due to angiogenesis in the choroidal area after RPE cell death 9 .The initiation of AMD is believed to involve the death of RPE cells, which can be caused by oxidative stress, inflammation, and carbonyl stress 31 .There are various reactive oxygen species (ROS), including hydroxyl radical (OH • ), hydroxide ion (HO -), triplet oxygen ( 3 O 2 ), superoxide anion (O 2 •− ), peroxide ion (O 2 2− ), hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO • ) 34 .The ROS related to AMD occurrence have been identified www.nature.com/scientificreports/as OH •35 , O 2 •−36 , and H 2 O 2 37 .However, the relationship between NO • levels and AMD occurrence is unclear, as controversial results exist, such as upregulation 38 and down-regulation 39 in AMD patients.Oxidative stress and inflammation are deeply related to the development of several pathologies 34 , as inflammatory cells stimulate oxidative stress 40 and ROS induce an increase in proinflammatory gene expression 41 .The Keap1/Nrf2 ARE is one of the representative anti-oxidative systems.It can control not only oxidative stress but also inflammation through the NF-κB pathway.This pathway is suggested as a good therapeutic target against many diseases, such as cancer, diabetes, inflammatory disease, and respiratory disease 42,43 .Carbonyl stress refers to the negative process in several parts of the bio-organism, such as protein, plasma membrane, and DNA, that is caused by carbonyl compounds generated through non-oxidative mechanisms 44 .Some carbonylation-related metabolites, such as 4-HNE 45 , trans-4-hydroxy-2-hexenal (4-HHE) 46 , and malondialdehyde (MDA) 47 , stimulate the occurrence of AMD.Apoptosis can be classified into extrinsic and intrinsic pathways 32 .The extrinsic pathway is initiated by the interaction of transmembrane receptors and ligands, such as tumor necrosis factor receptor 1 (TNFR1)/TNFα, FasR/FasL, and death receptors (DRs)/tumor necrosis-related apoptosis-inducing ligand (TRAIL) [48][49][50] .The intrinsic pathway is deeply connected to mitochondrial changes, which can be positively or negatively controlled by Bcl-2 family proteins but is not related to death receptors 51 .Bcl-2 family proteins include anti-apoptotic proteins, such as Bcl-2, Bcl-extra (Bcl-x), and Bcl-xL, and pro-apoptotic proteins, such as Bcl-2 associated X (Bax), Bcl-2 antagonist/killer 1 (Bak), and Bim.
The atrophic AMD is initiated by the death of RPE cells, which is caused by oxidative stress, inflammation, and carbonyl stress.Although there are many risk factors, such as aging, tobacco smoking, cataract surgery, family history, and overweight, the proximate and major factor is believed to be the epoxide type of A2E, which is produced by A2E and blue light 6 .However, it is unclear what level of A2E and intensity of blue light are required to induce an in vitro AMD model.From various studies, the level of A2E has been reported to range from 20 to 100 μM, and the intensity of blue light has been tested from 0.04 to 35 mW/cm 215-17 .To investigate similar conditions in bio-organisms with atrophic AMD, we evaluated the permeability of A2E to the cytoplasm and the relationship between RPE cell death and A2E level/blue light intensity (Figs. 3 and 4).A2E was found to pass through the cell membrane in a dose-dependent manner (Fig. 3A), and its cytotoxic effect on ARPE-19 cells increased with the level of A2E in the cytoplasm and the intensity of blue light (Fig. 3B).The population of apoptotic cells was increased in a dose-dependent manner with A2E and blue light treatment, but the number of necrotic cells also increased (Fig. 4A and Table 3).Taking into account both the number of apoptotic and necrotic cells, the combination of 30 μM A2E treatment and 20 mW/cm 2 blue light transillumination was selected as the best in vitro AMD model condition.To confirm the apoptosis-related oxidative stress and carbonyl stress pathway, several factors were analyzed.Under 30 μM A2E treatment and 20 mW/cm 2 blue light transillumination, the Nrf2/HO-1 anti-oxidative system was suppressed and the level of 4-HNE was increased in ARPE-19 cells (Fig. 4B).These results indicate that ARPE-19 cell death is induced by Nrf2/HO-1 anti-oxidative system-related oxidative and carbonyl stress (Figs.S1, S3, S3) .
While developing potential synthetic chemicals against atrophic AMD is an option, natural products that have been used as culinary and/or medicinal materials for a long time can be another alternative, as they may be relatively safe if they have been consumed as edible substances for a prolonged period.However, before considering them as candidates, their therapeutic mechanisms, such as anti-oxidation, anti-inflammation, and/ or anti-carbonylation for preventing RPE cell death, should be confirmed.C. lanceolata is a culinary/medicinal material that has been used for a long time in Asia and has been reported to have biological effects, such as anti-hepatitis 24 , anti-inflammatory 25 , and anti-asthmatic effects 26 .These observations suggest that C. lanceolata has anti-inflammatory and immune modulation effects.In this study, we evaluated the anti-apoptotic effect of C. lanceolata on RPE cells by analyzing apoptosis-related factors, such as oxidative and carbonyl stress.C. lanceolata dose-dependently decreased the level of apoptosis in ARPE-19 cells induced by A2E and blue light co-treatment (Fig. 5B & Table 4).When comparing the anti-apoptotic potency between C. lanceolata and lutein (positive control drug), the normal cell population in 25 μM lutein treatment was 82.2 ± 0.95% and in 25 μg/mL C. lanceolata treatment was 83.5 ± 2.03%.As the comparison of 25 μM lutein and 25 μg/mL C. lanceolata treatment did not indicate a statistically significant difference, the anti-apoptotic potency of C. lanceolata was assumed to be similar to that of lutein.Based on its anti-apoptotic potency, we assessed the involvement of the Bcl-2 familyrelated intrinsic apoptosis pathway, the Keap1/Nrf2 anti-oxidative system, and carbonyl stress (Fig. 6).As shown in Fig. 5A, C. lanceolata effectively modulated the Bcl-2 family by increasing the levels of anti-apoptotic Bcl-2 proteins, such as Bcl-2 and Bcl-XL, while decreasing that of the pro-apoptotic protein, Bim. C. lanceolata not only stimulated the Keap1/Nrf2/HO-1 ARE pathway (Fig. 6B-D) but also inhibited the expression of 4-HNE (Fig. 6B).

Conclusion
Based on these results, we obtained two findings.First, we found that the appropriate condition for an in vitro model of AMD is the treatment of ARPE-19 cells with 30 μM A2E and transillumination with 20 mW/cm 2 blue light.This combination effectively induced oxidative and carbonyl stress, leading to the death of ARPE-19 cells.Second, we found that C. lanceolata shows promise as an anti-AMD agent.It effectively suppressed cellular stresses through Nrf2/HO-1 ARE activation and 4-HNE suppression, ultimately preventing apoptosis by deactivating the intrinsic apoptosis pathway.

Figure 1 .
Figure 1.N-retinylidene-N-retinyl-ethanolamine (A2E) synthesis and the light intensity of the manufactured blue light transilluminator.(A) The scheme of A2E synthesis.(B) The result of A2E synthesis.Synthetic A2Es consisted of one A2E and two A2E isotypes.(C) The intensity of the blue light transilluminator.The peak wavelength of the irradiated light was 454.6 nm, with the majority of light falling within the range of blue light.

Figure 2 .
Figure 2. Cell culture protocols depending on the analysis and sample harvest time.

Figure 4 .
Figure 4. Apoptosis, oxidative stress, and carbonyl stress in ARPE-19 cells depending on the level of A2E and the intensity of blue light.(A) The results of the TUNEL assay for ARPE-19 cell death at various doses of A2E and/or blue light intensities.Scale bar, 50 μm.Magnification, 1000 × .(B) The results of apoptosis-related pathway analysis.N = 3.

Figure 6 .
Figure 6.Codonopsis lanceolata regulated the apoptosis of ARPE-19 cells through the activation of the Keap1/ Nrf2/HO-1 pathway and the suppression of 4-HNE.(A) C. lanceolata modulated the Bcl-2 family, increasing the levels of the antiapoptotic proteins Bcl-2 and Bcl-XL, while decreasing the level of the proapoptotic protein Bim (N = 3).(B) C. lanceolata treatment activated the anti-oxidative Keap1/Nrf2/HO-1 pathway but decreased the expression of 4-HNE in a dose-dependent manner (N = 3).(C) C. lanceolata treatment stimulated the expression of nuclear Nrf2, which functions as a transcription factor for HO-1 (green spots).(D) C. lanceolata treatment dose-dependently induced the expressions of Keap1 and HO-1.Scale bar, 50 μm.Magnification, 1000 × .